The homeostatic mechanisms underlying the absence of vasculature (blood and lymphatic vessels) in the human and mouse cornea are remarkably intriguing given the highly vascularized nature of the neighboring tissues, such as the ocular conjunctiva. This avascular disposition makes the cornea an important angiogenesis assay platform (Gimbrone et al., J. Exp. Med., 136(2): p. 261-76, 1972) allowing scientists to study the pro- and/or anti-angiogenic effects of several compounds in vivo. More than serving as the basis for an angiogenesis assay model, the phenomenon of corneal avascularity serves essential physiological functions. Corneal neovascularization precludes optimal vision and compromises corneal immunological privilege.
In 1905, ophthalmologist Edward Zim performed the first corneal transplant in a human subject (Moffatt et al., Clin. Experiment. Opthalmol., 2005.33(6): 642-57, 2005). Since then, corneal transplants have become the most common type of solid organ transplantation in the world. Nearly 46,000 corneal transplants are performed yearly in the United States. In addition to being the most prevalent, corneal allograft transplantation is also the most successful intervention among other commonly transplanted organs. However the long-term outcome of this intervention is greatly influenced by pre-operative risk factors, with corneal neovascularization (high-risk group) being an important negative predictor of corneal allograft survival. While graft survival is approximately 90% in the low-risk group (no pre-operative inflammation or neovascularization) these numbers drastically fall to roughly 35% in the high risk group (Williams et al., Transplant. Proc., 29(1-2): 983, 1997).
While the absence of blood and lymphatic vessels in the cornea is known to play a critical role in maintaining its immune privilege (Cursiefen et al., Cornea, 22(3): 273-81, 2003), other immune-protective mechanisms have been described. One such mechanism is referred to as anterior chamber-associated immune deviation (ACAID). ACAID is regarded as the ability of antigen presenting cells and antigens from anterior chamber associated tissues (i.e., cornea) to directly enter the blood circulation through the trabecular meshwork, homing to the spleen where immune tolerance is induced (Wilbanks et al., Immunology, 71(4): 566-72, 1990; Wilbanks et al., Immunology, 71(3): 383-9, 1990; Niederkorn et al., Invest. Opthalmol. Vis. Sci., 37(13): p. 2700-7, 1996). Additionally, tissues from the anterior segment of the eye have been reported to express a protein named Fas-ligand which induces apoptosis in activated immune cells (Fas-receptor positive) (Griffith et al., Science, 270(5239): 1189-92, 1995), thus protecting the cornea from damage by activated lymphocytes. These mechanisms are thought to collectively down-regulate inflammation in the cornea therefore preserving corneal clarity which is essential for optimal vision.
Major advances in the study of corneal lymphangiogenesis have taken place since the discovery of VEGFR-3 and its ligands VEGF-C and VEGF-D (Kaipainen et al., Proc. Natl. Acad. Sci. U.S.A., 92(8): 3566-70, 1995; Joukov et al., Embo. J., 15(2): 290-98, 1996; Achen et al., Proc. Natl. Acad. Sci. U.S.A., 95(2): 548-53, 1998). The identification of specific cellular markers preferentially expressed by lymphatic endothelial cells, such as LYVE-1 (Banerji et al., J. Cell. Biol., 144(4): 789-801, 1999), Prox1 (Wigle et al., Cell, 98(6): 769-78, 1999) and podoplanin (Breiteneder-Geleff et al., Am. J. Pathol., 151(4): 1141-5212, 1997), have also propelled great advances to the field of lymphangiogenesis. The growth of lymphatic vessels into the cornea generally occurs after corneal injury and inflammation, which in turn is associated with increased levels of VEGF-C (Jiang et al., J. Huazhong Univ. Sci. Technolog. Med. Sci., 24(5): 483-5, 2004; Kure et al., Invest. Opthalmol. Vis. Sci., 44(1): 137-44, 2003). The newly formed lymphatic vessels are thought to permit an outwards route through which corneal transudate and APCs are carried from the interstitial space into the lymphatic system and later back into the blood circulation. This drainage pathway becomes extremely deleterious in the context of corneal transplantation. Under these circumstances, the alternative route bypassing the standard outflow pathway (i.e. trabecullar meshwork in the anterior chamber) allows for antigens from the donor cornea to escape through the lymphatic system and into the draining lymph node where a graft rejection reaction is initiated (Yamagami et al., Cornea, 21(4): 405-9, 2002; Liu et al., J. Exp. Med., 195(2): 259-68, 2002). By targeting corneal angiogenesis with VEGF-A binding molecules (VEGF-trap), Cursiefen et al. demonstrated that allograft survival was inversely related to the amount of neovascularization in the murine corneal transplantation model. The significance of this alternate drainage pathway to corneal alloimmunity and graft rejection has also been portrayed in a study showing that removal of cervical lymph nodes significantly increased the graft survival rates in the low and high-risk groups (Yamagami et al., Cornea, 21(4): 405-9, 2002; Yamagami et al., Invest. Opthalmol. Vis. Sci., 42(6): 1293-8, 2001).
The surgical procedures used in corneal allograft transplantation require very delicate techniques to prevent adverse inflammatory reactions which may compromise outcome. The corneal graft is initially attached to the recipient's ocular surface with the placement of small sutures. Paradoxically, in a vastly employed injury animal model of corneal angiogenesis, similar intrastromal sutures are used as a method of eliciting blood and lymphatic vessel growth (Sonoda et al., Cornea, 24(8 Suppl): S50-S54, 2005). Because suture placement is a requirement for corneal transplantation as well as a pro-angiogenic stimulus, it becomes necessary to dissect the molecular mechanisms modulating the growth of blood and lymphatic vessels under these circumstances.
Vasculogenesis relates to the embryological and/or post-natal development of vasculature from bone-marrow derived endothelial precursor cells (EPC), whereas angiogenesis is a biological process that denotes the formation of vascular tissue from pre-existing vessels (Asahara et al., Science, 275(5302): 964-7, 1997). Functionally, angiogenesis may be subcategorized as hemangiogenesis, the growth of blood vessels; and lymphangiogenesis, which stands for the emergence of lymphatic vessels.
The VEGF family of molecules is thus far the most studied modulators of angiogenesis. This family of molecules includes VEGF, also known as VEGF-A, placental growth factor (PLGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. The pro-angiogenic effects of these growth factors are primarily mediated by binding and activation of their cognate receptors (VEGFRs). While VEGF-A is capable of binding and activating VEGFR-1 and VEGFR-2 (Ferrara et al., Nat. Med., 9(6): 669-76, 2003), VEGF-C and VEGF-D signal through VEGFR-3 and VEGFR-2 (Adams et al., Nat. Rev. Mol. Cell. Biol., 8(6): 464-78, 2007). VEGF-B and PLGF bind exclusively to VEGFR-1 and likewise, VEGF-E binding is restricted to VEGFR-2. It is important to note that VEGFR-1 and VEGFR-2 are primarily expressed in blood endothelial cells whereas VEGFR-2 and VEGFR-3 are mainly expressed in lymphatic endothelial cells (Karkkainen et al., Nat. Cell. Biol., 4(1): E2-5, 2002. This is important given that VEGF-A largely drives hemangiogenesis while VEGF-C mediates lymphangiogenesis.
VEGFRs are tyrosine kinase-type receptors (RTK) that belong to the immunoglobulin (Ig) superfamily of molecules. As such, they are comprised of 7 Ig-like domains in their extracellular segment, a transmembrane domain and an intracellular tyrosine kinase domain. The intracellular signaling cascade that follows VEGFRs activation is very complex and finely orchestrated. Several intracellular messenger systems become activated (i.e., PKC, PI3K, Src, MAPK) ultimately resulting in endothelial cell migration, proliferation, increased survival (i.e., anti-apoptosis) and increased vascular permeability (Ferrara et al., Nature, 438(7070): 967-74, 2005).
The imperative significance of VEGFs signaling to vasculogenesis and angiogenesis was made evident by the observation that the deletion of vegf-a, vegfr-1, vegfr-2 and vegfr-3 genes gave rise to lethal phenotypes that transpired at early embryonic stages. Abnormal blood vessel development and lethality were observed when inactivating the vegf-a gene in two independent studies (Carmeliet et al., Nature, 380(6573): 435-9, 1996; Ferrara et al., Nature, 380(6573): 439-42, 1996). The targeted deletion of vegfr-1 was associated with abnormal formation of blood vessel channels (Fong et al., Nature, 376(6535): 66-70, 1995), whereas abrogation of vegfr-2 resulted in failure of blood island formation (Shalaby et al., Nature, 376(6535): 62-6, 1995). VEGFR-3 deletion was also lethal and associated with aberrant development of large vessels and cardiac failure due to pericardial fluid accumulation (Dumont et al., Science, 282(5390): 946-9, 1998).
Site-directed mutagenesis studies (Wiesmann et al., Cell, 91(5): 695-704, 1997) and bioengineering of mosaic molecules (Jeltsch et al., J. Biol. Chem., 281(17): 12187-95, 2006) have unveiled critical ligand binding domains for VEGF-A and VEGF-C to their cognate receptors. Ig-like domain 2 of VEGFR-1 and Ig-like domains 2 and 3 of VEGFR-2 are critical for VEGF-A binding. VEGF-C, on the other hand, requires only Ig-like domain 2 of VEGFR-2 and Ig-like domain 1 and 2 of VEGFR-3.
A soluble splicing variant of VEGFR-1 (sVEGFR-1 or sFLT-1) was first described by Kendall and Thomas (Proc. Natl. Acad. Sci. U.S.A., 90(22): 10705-9, 1993). This isoform receptor is comprised of the first 6 of the 71 g-like domains normally present in the extracellular segment of the membrane bound VEGFR-1. The alternative splicing event that gives rise to this soluble isoform takes place in the junction between exon 13 and intron 13/14 of VEGFR-1 pre-mRNA. In this case, intron 13/14 becomes part of exon 13 and due to the presence of an in-frame stop-codon, a truncated (hence soluble) protein is instead produced. sVEGFR-1 therefore has a unique c-terminus that includes 31 amino acids. Since the critical VEGF-A binding domain of VEGFR-1 is conserved in the alternate soluble protein, it avidly binds VEGF-A (Kendall et al., Proc. Natl. Acad. Sci. U.S.A., 90(22): 10705-9, 1993). The absence of the transmembrane domain and tyrosine kinase domains precludes receptor signaling and sVEGFR-1 is considered an endogenous anti-angiogenic molecule. Alternative splicing mechanisms similar to that of sVEGFR-1 are not at all uncommon. In fact, comparable splicing events are responsible for the generation of several other soluble variants derived from membrane bound proteins, such as, the alpha subunit of interleukin-5 (IL-5) receptor (Tavernier et al., Proc. Natl. Acad. Sci. U.S.A., 89(15): 7041-5, 1992), immunoglobulin heavy chain (Peterson, Immunol. Res., 37(1): 33-46, 2007), fibroblast growth factor receptors (Johnson et al., Mol. Cell. Biol., 11(9): 4627-34, 1991; Werner et al., Mol. Cell. Biol., 12(1): 82-8, 1992), and neuropilin-1 (Gagnon et al., Proc. Natl. Acad. Sci. U.S.A., 97(6): 2573-8, 2000).
Since its discovery in 1993, soluble VEGFR-1 has been extensively studied and implicated in several pathological states including pre-eclampsia (Tsatsaris et al., J. Clin. Endocrinol. Metab., 88(11): 5555-63, 2003), sepsis (Tsao et al. Crit. Care Med., 2007), arthritis (Afuwape et al., Gene Ther., 10(23): 1950-60, 2003) and cancer (Elkin et al., J. Natl. Cancer Inst., 96(11): 875-8, 2004). In the cornea, it has been shown to exert a critical anti-angiogenic function. sVEGFR-1 is a key modulator of corneal avascularity, especially due to the presence of VEGF-A in the normal uninjured cornea (Ambati et al., Nature, 443(7114): 993-7, 2006).